Potent inhibition of influenza virus by specifically designed short interfering RNA

ABSTRACT

This patent application discloses siRNA sequences against the constant region of the influenza virus nucleoprotein gene comprising:  
                         Sense strand:             5′ UGAAGGAUCUUAUUUCUUCdTdT 3′   (SEQ ID NO: 1)           Anti sense strand:     3′ dTdTACUUCCUAGAAUAAAGAAG 5′   (SEQ ID NO: 2)           Sense strand:     5′ UGAAGGAUCUUAUUUCUUCGGdTdT 3′   (SEQ ID NO: 3)           Anti sense strand:     3′ dTdTACUUCCUAGAAUAAAGAAGCC 5′   (SEQ ID NO: 4)           Sense strand:     5′ GGAUCUUAUUUCUUCGGAGACdTdT 3′   (SEQ ID NO: 5)           Anti sense strand:     3′ dTdTCCUAGAAUAAAGAAGCCUCUG 5′   (SEQ ID NO: 6)                              
said sequences being inhibitory against influenza virus in animals including humans. The invention further includes one or more of said siRNA sequences in the form of an aqueous suspension suitable for nasal inhalation. Still further, the invention includes one or more of said siRNA sequences in the form of a plasmid expressing intracellularly in animals including humans. In another aspect, the invention includes one or more of said siRNA sequences in the form of an AAV vector adapted to express intercellularly and establish a permanent inhibitory effect against influenza virus by integrating to the cellular chromosome of animals including humans. The invention also includes the administration to an animal including humans, in a therapeutically effective amount, at least one of said siRNA sequences.

This patent claims the filing date of U.S. Provisional PatentApplication Ser. No. 60/687,373, filed Jun. 3, 2005, the disclosures ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Viruses are packets of infectious nucleic acid surrounded by protectivecoats which lack metabolic energy due to the absence of independentmetabolism, and are incapable of growth by protein synthesis orreproduction apart from living cells. They have a prokaryotic geneticapparatus and usually contain either DNA or RNA, but not both and areusually covered by a protein shell or capsid which protects the nucleicacid.

The influenza or Orthomyxoviridae viruses cause the common influenza andinfluenza-like infections in humans and other mammals. These virusescontain negative single-stranded RNA as the genetic material and usuallyin eight segments. Included are influenza types A, B, and C as well asavian flu virus (H₅N1).

The influenza virus infects the respiratory tracts of millions of peopleevery year and is the cause of about 20,000 deaths annually in the US.The remarkable success of the influenza virus is due to genusOrthomyxoviridae's ability to undergo genetic reassortment to produceantigenic shift among its three types (species), A, B, and C, and withinsubstrains. The genome of the Orthomyxoviruses is typified by eightsegments of single-strand negative-sense RNA protected within anucleocapsid structure. Each of the eight segments of thesingle-stranded negative-sense RNA codes for a particular viral protein:virion surface glycoproteins, hemagglutinin and neuraminidase forattachment process to the host cell surface receptor; matrix 1 andmatrix 2 proteins for ion channel; PA, PB1, and PB2 for transcriptaseand replicase enzymes for transcription and replication of viral genome;nucleocapsid, NP protein, for protection of all the viral genomesegments from degradation by host RNase (FIG. 1). This nucleocapsid genehas a highly conserved region, and therefore, holds promise as a targetagainst which an effective antiviral strategy can be developed.

RNA interference (RNAi) is a recently discovered and developed antiviralstrategy in which gene silencing is effected by homologous short (21-23bp) dsRNA fragments known as short interfering or siRNA. When a longdsRNA is introduced into a cell line, the cellular enzyme called Dicerwill cleave it into short interfering RNA (siRNA) molecules. This shortinterfering RNA molecule is now called the guided RNA. The guided RNAwill guide the RNA-lnduced-Silencing-Complex (RISC) to the homologoustarget mRNA. Once it forms a hybrid structure to the homologous mRNA,the RISC will cleave the mRNA. As a result, protein that is encoded bythe mRNA will no longer be produced and this will cause the silencing ofthe gene.

A recently published patent application, United States PatentApplication Publication No. US 2004/0242518 A1 to Chen et al., publishedDec. 2, 2004, discloses a siRNA (FIG. 21A, NP 1496) which partiallyoverlaps the siRNA of this invention and cleaves at a different targetnucleotide.

SUMMARY OF INVENTION

Briefly, this invention comprises a siRNA sequence against the constantregion of the influenza virus nucleoprotein gene comprising: Sensestrand: 5′ UGAAGGAUCUUAUUUCUUCdTdT 3′ Anti sense strand: 3′dTdTACUUCCUAGAAUAAAGAAG 5′said sequence being inhibitory against influenza virus in animalsincluding humans.

The two longer siRNA sequences, while useful, were found to havesomewhat lesser efficacies were: a) Sense strand: 5′UGAAGGAUCUUAUUUCUUCGGdTdT 3′ Anti sense 3′ dTdTACUUCCUAGAAUAAAGAAGCC 5′strand: and b) Sense strand: 5′ GGAUCUUAUUUCUUCGGAGACdTdT 3′ Anti sense3′ dTdTCCUAGAAUAAAGAAGCCUCUG 5′ strand:Both these siRNA preparations were 23 bp in length.

The invention further includes one or more of said siRNA sequences inthe form of an aqueous suspension suitable for nasal inhalation.

Still further, the invention includes one or more of said siRNAsequences in the form of a plasmid expressing intracellularly in animalsincluding humans.

In another aspect, the invention includes one or more of said siRNAsequences in the form of an AAV vector adapted to expressintercellularly and establish a permanent inhibitory effect againstinfluenza virus by integrating to the cellular chromosome of animalsincluding humans.

In yet another aspect, the invention also includes the administration toan animal including humans, in a therapeutically effective amount, atleast one of said siRNA sequences.

The objective of this study is to develop an antiviral therapeuticutilizing siRNA technology to target the constant region of theinfluenza virus nucleocapsid gene. Since influenza virus is a negativess RNA virus, our specifically designed siRNA against NP gene would havedual inhibitory effect. The sense strand of the siRNA would directlycleave the viral genome and the anti-sense strand would target andcleave the homologous mRNA causing post transcriptional gene silencing.

Strategy to Design and Develop the siRNA Against Influenza Virus NP Gene

Influenza is an enveloped virus with segmented single stranded negativeRNA as the genetic material. The influenza virus contains eight segmentsor seven segments of (−) single stranded RNA genomes depending on twogenera: influenza A and B viruses, and influenza C virus. Influenza Aand B contain eight segments (−) of single stranded RNA genomes whereasthe influenza C virus contains only seven (−) single strands of RNA asgenome. The major distinction between influenza A, influenza B, andinfluenza C are the antigenic differences between their nucleoproteins(NP) and matrix proteins (M). The NP is the major structural proteinthat interacts with all the viral RNA segments, forming the viralribonucleoprotein (RNP) complex.

The virology community tends to focus almost exclusively on two targetsin the viral genome, the neuraminidase and hemoglobinase genes. It hasbeen observed that an alternative gene coding for the NP proteinpossesses characteristics that suggest a greater suitability as a drugtarget. Neuraminidase and hemagglutinin, for instance, are known to bevariable across different strains of flu. Therapeutics designed totarget one form of neuraminidase gene or its protein product, therefore,might not be effective against a flu strain with a different form of thegene. Certain NP gene sequences, however, appear to be more highlyconserved across different strains of flu, thereby permitting for thepossibility of therapeutics targeting the gene or its protein to beeffective against a broad spectrum of strains. In addition, the NP geneplays an extremely important role in the survival of the influenza virusby protecting all of its segments of the viral genome. A therapeuticsubstance diminishing or eliminating the NP gene or its proteinproduct's ability to protect the viral genome should therefore leave thevirus more susceptible to attack by the host's defenses.

Both the viral (−) strand RNA and the template (+) strand RNA areclosely associated with the nucleocapsid; however, the mRNA (+) strandsare not encapsidated. This close association of the NP to the viral RNAis to encapsidate the viral genome and perhaps protect it from the hostRNase. Furthermore, it appears that NP is one of the most importantproteins in the influenza virus structure since the virus must formribonucleoprotein (RNP) complexes with all the genomic RNA segments.Therefore, it is likely that the best antiviral strategy is to targetthe NP gene of the influenza virus. Since influenza virus genomesundergo considerable changes, the siRNA of this invention was designedencompassing the conserved region of NP gene of influenza virus.

THE DRAWINGS

An enlarged drawing of influenza virus is shown in FIG. 1. This drawingis generic to influenza types A, B and C.

An enlarged view of influenza virus RNP is shown in FIG. 2.

FIG. 3 is a bar graph further explained in Example 1.

FIG. 4 is a bar graph also explained in Example 1.

FIG. 5 is a bar graph further explained in Example 2.

FIG. 6 is a bar graph explained in Example 3.

DESCRIPTION OF PREFERRED EMBODIMENTS

From the GeneBank database, the NP gene sequence of more than 20different influenza virus isolates was aligned and the non-variableregion of these sequences (nucleotides 1491-1538) identified. From thesecondary structural analyses of this region of the viral genome, thesequence of the siRNA which has been found to be inhibitory of theinfluenza virus in animals including humans was identified.

The siRNA sequences of this invention, 19 to 23 nucleotides long, arebased on the NP gene. The first in vivo experiment entailed dosinghealthy mice (Balb/c) with plasmids or siRNA vectors containing the leadsiRNA therapeutic (19 nucleotides long). After 24 hours, the mice werechallenged with strains of murine flu virus (X88 and PR8) normallyconsidered to be less pathogenic, but in this case, engineered toexhibit a high degree of virulence. The exposed mice were weighed todetermine any loss of body mass, an endpoint used in place of lethality.It was found that even small doses prevented signs of infectionthroughout the two to three weeks of the study.

In a separate experiment heathy mice were infected with lethal doses ofinfluenza virus and after 24 hours they were treated with plasmidexpressing siRNA. It was found that this therapeutic approach alsoprotected all the experimental mice.

The NP gene regions of avian flu were also compared with the siRNAsequences of this invention. Among the 50 isolates of H5N1 studied, all50 showed 100% homology with the NP regions corresponding to the siRNAof this invention, suggesting they would be effective against avian flu.

Development of Different Forms of siRNA

The preferred siRNA of this invention was developed in three forms:

-   -   (1) Twenty one nucleotide sense and antisense RNA in duplex form        having the sequence first identified above. This siRNA was        synthetically made. The siRNA can be administered readily        internasally in the form of a mist by inhalation. This results        is an effective but relatively short useful life in the order of        days. The mist is made up of the siRNA in the form of a cationic        polymer (polyelhylenimine) medium to provide a suspension.    -   (2) Plasmid expressing sense and anti-sense strands of the same        siRNA under the influence of pol III promoter, U6+1. The        promoter and the sequence encoding the sense strand followed by        a TTTTT terminator, and the promoter, the sequence encoding the        anti-sense strand of siRNA followed by TTTTT terminator was        cloned into plasmid pcDNA4/TO. The plasmid expressing siRNA can        also be administered intranasally in the form of a cationic        polymer emulsion. The inhibitory effect produced by the plasmid        has a longer life than the synthetic siRNA described in (1).    -   (3) AAV vector expressing sense and anti-sense strands of the        same siRNA under the influence of pol III promoter, U6+1. The        AAV vector is a benign virus vector which enters the cell of the        host and integrates into the cellular chromosome, and expresses        siRNA constitutively to yield a permanent inhibitory effect        against influenza virus in the host.

This invention provides novel short-interfering RNA (siRNA) sequencestargeting a highly conserved region of the influenza viral genome. Themost effective siRNA target sequence was found to be within thenucleoprotein gene, one of the eight genes comprising the influenzagenome. The preferred siRNA, 19 base-pairs long, was evaluated byintroducing it into mice later infected with a murine flu virus. Weightloss, was used as the primary endpoint of the studies. Weight loss wasnot observed in the experimental infected mice.

The following Examples are illustrative:

EXAMPLE 1

Graphs 3 and 4 demonstrate the efficacy of siRNA in commerciallyavailable MDCK cell cultures. The vertical axis indicates the level ofinfluenza virus. The horizontal axis indicates time (hours). The lineindicated as blue shows the results over time for a control in which nosiRNA is added to the cell culture. The line indicated in red shows theresults over time when the synthetic si RNA in aqueous suspension wastransfected to the cell culture. The line indicated in green shows theresults over time when siRNA in plasmid form was transfected to the cellculture. Twenty four hours after transfection cells were infected withinfluenza virus.

Graph 4 presents the results shown in Graph 3 in bar form and shows theinhibitory effect of siRNA in synthetic and plasmid form when comparedto the control.

EXAMPLE 2

Graph 5 shows the results conducted in a standard laboratory mouse(Balb/c) model. The vertical axis expresses the average body weight infive (5) mice. The horizontal axis is time expressed in days. The lineindicated in blue shows the effect on the average weight of five miceadministered internasally with the cationic lipid emulsion of the siRNAplasmid in a 19 μg/mouse dosage. The red line shows the results when thesame emulsion was identically administered to five mice in a 7 μgdosage. In both cases, the dosage was administered at day “0”.Thereafter, no further siRNA plasmid was given. At day “1”, a lethaldose of influenza virus was given to both groups of mice. The effect onbody weight was taken as a measure of efficacy. It is significant thatat the higher dosage (blue line), the siRNA plasmid was almostcompletely effective in preventing loss of body weight, indicating thatthe siRNA plasmid is silencing the influenza virus nucleocapsid gene.

EXAMPLE 3

In Graph 6, the same mouse model was used. The brown line was a controlgroup of five mice which received neither siRNA in any form nor was thisgroup exposed to influenza virus. The line indicated as green shows theresult with five mice who intranasally received siRNA in the AAV-siRNAform on day “0” and a lethal dose of the influenza virus at day “1”. Theline indicated as blue represents the results in five mice whichreceived no siRNA in any form but did receive the influenza virus on day“1”. As can be seen, the control (brown line) group continued to thriveand in fact continue to survive long past the end of the brown line, aswas to be expected. The interesting results are seen when comparing thegreen and blue indicated lines. The influenza virus quickly killed allof the mice which had not received the siRNA in any form (blue indicatedline). However, the mouse group receiving the AAV-siRNA followed by alethal dose of influenza virus 24 hours later (green line) continued tothrive. Further, in Graph 4, the mouse group indicated by the green linewas subsequently exposed to a doubled dosage of influenza virus on day13 continued to thrive. Subsequently, the influenza virus dosage wasdoubled again on day 23 and the same five mice continued to thrive, thusindicating that the AAV-siRNA had established a permanent inhibitoryeffect against the influenza virus in these mice.

The invention consists of, consists essentially of, and comprises theabove nucleotide sequences in all forms in which said sequences are thetherapeutically active agent. Normally, the sequences are isolated andpurified and used in pharmaceutical carriers, or incorporated intoplasmids or vectors. The techniques for the preparation of suchpharmaceutical compositions, the incorporation of the nucleotidesequences into plasmids and vectors can be carried out by those ofordinary skill in this art.

In regard to the effective therapeutic dose, it is somewhat difficult toascertain at this time. However, from the mouse experiment with plasmidform of siRNA and challenge with a lethal dose of influenza virus, weestimate about 2-3 μg/kg body weight may be the effective therapeuticdose against the influenza virus infection in animals including humans.The same dosages are deemed effective for the vector form. The siRNAsuspension is also useful around this dosage range.

1. A siRNA sequence against the constant region of the influenza virus nucleoprotein gene comprising: Sense strand: 5′ UGAAGGAUCUUAUUUCUUCdTdT 3′ (SEQ ID NO: 1) Anti sense strand: 3′ dTdTACUUCCUAGAAUAAAGAAG 5′ (SEQ ID NO: 2)

said sequence being inhibitory against influenza virus in animals including humans.
 2. A siRNA sequence against the constant region of the influenza virus nucleoprotein gene comprising: Sense strand: 5′ UGAAGGAUCUUAUUUCUUCGGdTdT 3′ (SEQ ID NO: 3) Anti sense strand: 3′ dTdTACUUCCUAGAAUAAAGAAGCC 5′ (SEQ ID NO: 4)

said sequence being inhibitory against influenza virus in animals including humans.
 3. A siRNA sequence against the constant region of the influenza virus nucleoprotein gene comprising: Sense strand: 5′ GGAUCUUAUUUCUUCGGAGACdTdT 3′ (SEQ ID NO: 5) Anti sense strand: 3′ dTdTCCUAGAAUAAAGAAGCCUCUG 5′ (SEQ ID NO: 6)

said sequence being inhibitory against influenza virus in animals including humans.
 4. At least one of the siRNA sequences of claims 1, 2 or 3 in the form of an aqueous suspension suitable for nasal inhalation.
 5. At least one of the siRNA sequences of claims 1, 2 or 3 in the form of a plasmid expressing intracellularly in animals including humans.
 6. At least one of the siRNA sequences of claims 1, 2 or 3 in the form of an AAV vector adapted to express intercellularly and establish a permanent inhibitory effect against influenza virus by integrating to the cellular chromosome of animals including humans.
 7. The administration to an animal including humans of a therapeutically effective amount of at least one of the siRNA sequences of claims 1, 2 or
 3. 8. The method of claim 7 wherein the administration is by nasal inhalation in the form of an aqueous mist.
 9. The method of claim 7 wherein the administration is in the form of a plasmid.
 10. The method of claim 7 wherein the administration is in the form of a AAV vector.
 11. The method of claim 7 wherein the administration is in the form using plasmid pcDNA4/TO.
 12. The method of claim 7 wherein the administration is effective against influenza virus A, B or C.
 13. The method of claim 7 wherein the administration is effective against avian influenza (H5N1). 